Microsomes are subcellular fractions containing membrane fragments. In vitamin E-deficient rats, microsomes are especially prone to oxidative free radical damage. This can be quantified in terms of the production of thiobarbituric acid reactive substances (TBARS) which result from radical-mediated destruction of the polyunsaturated fatty acid constituents. Consequently, this is a useful biological model to determine the efficacy of phytochemicals as antioxidant membrane protectants. Vitamin E-deficient microsomal suspensions were incubated for 30 minutes with one of myricetin, sample A, sample B, sample C (as shown below) or d-alpha-tocopherol, or with a compound 9c, 9d, 9e, 9e*, 9f, 9g, 9g*, 9h, 9i* or 9j (prepared as described above in Examples 1 to 10).

The microsomal suspension was then added to solutions containing Fe(II)-ADP/ascorbate to initiate free radical-mediated oxidation and incubated for a further 0, 5, 10, 15 or 20 minutes. TBARS production was then measured by HPLC.
In all the following examples and discussions, we will use the traditional numbering scheme for flavonoids rather than that defined in Formula 1 above. The traditional numbering is as shown below:
Results
In the absence of antioxidant protection (−E), TBARS production increases with time. Myricetin (M), although a potent antioxidant in chemical systems affords almost no protection. Control B, in which the two hydroxyls of myricetin have been removed to increase lipophilicity, is very soluble in octanol, and we have shown by ESR that it retains potent antioxidant activity. However, it does not give rise to significant membrane protective effects. Replacing the B ring hydroxy groups with methoxy produces a non-protective compound which has a lack of antioxidant activity in the ESR chemical medical system. Control E, which comprises an unbranched alkyl chain linked to the A-ring via oxygen and with a C12 alkyl chain length, shows efficacy in the initial stages of microsomal oxidation. However, the protection is lost after 20 minutes. The target compounds according to the invention suppress oxidative damage throughout the 20 minute period and are comparable in effectiveness to dα-tocopherol (α).
Table 2 below gives the TBARs data obtained for compounds of varying chain length after 20 minutes incubation and nomalised to a tocopherol reading of 20. The higher the reading the lower the protection provided. The TBARS data for membrane protection versus compound are presented as bar graphs in FIG. 2a and FIG. 2b. The same TBARS data for membrane protection plotted against compound lipophilicity are presented as scatter plots in FIG. 3a and FIG. 3b, respectively.
Table 3 summarises the TBARs data obtained after 20 minutes incubation and normalised to a tocopherol reading of 20, for compounds having different head groups and chain substitution sites.
The data in FIG. 2a shows that for a given head group and position of attachment of the chain, cell membrane protection depends strongly on the chain length. The optimum chain length for a chain attached at the 7-position is in the range C6 to C12. The data in FIG. 3a shows that for a given head group and position of attachment of the chain, cell membrane protection depends strongly on the lipophilicity as represented by calculated ClogP values. For compounds 9 bearing a chain attached to the 7-position good membrane protection is afforded by compounds with ClogP values in the range 4 to 10 (the compound with a ClogP value of 12 is α-d-tocopherol). The data in FIGS. 2b and 3b show the effect of varying the site at which the chain is attached, of varying the head group and of varying the nature of the atom linking the chain to the head group. Compounds 9g, 11g, and 12 have the same head group and almost identical lipophilicities (ClogP values) but different membrane protecting properties. Thus, we argue that there is an orientation effect that means that there is an optimum chain length for a particular site of attachment of the chain to a particular head group. Compounds 9g, 13g and 15g have the same chain length and site of attachment of the chain. They also have the same number of hydroxyl groups attached to the B and C rings. It is clear that the substitution pattern on the B-ring affects cell membrane protection. In particular a 3,3′,4′,5′-tetrahydroxy-flavone head group as in compound 9g and a 3,2′,4′,5′-tetrahydroxy-flavone head group as in compound 13g give good membrane protection. The poor membrane protection exhibited by compound 15g may be the result of poor orientation as this may be affected by the head group. Comparing the data for compound Control E and compound 9h shows that when the chain is attached to the head group by an oxygen atom rather than a carbon atom, membrane protection is less. This may also be an orientation effect.
The length of the RA chain also appears to have a major impact on activity (see compounds 9j, 9h, 9g and 9d). The order of activity is C18≈C2<C12<C10. This is also reflected in the two branched chain compounds (9i* and 9g*), where the compound having C8 backbone has significantly higher inhibiting effects.
TABLE 1ReactionSubstitution PatternCompoundk2Stoichiometry34572′3′4′5′Catechin1574 ± 79 2.96 ± 0.01—H, —OH—H, —H—OH—OH—OH—OHTaxifolin337 ± 322.82 ± 0.05—H, —OH═O—OH—OH—OH—OHHesperitin  6 ± 0.50.20 ± 0.02—H, —H═O—OH—OH—OH—OMeApigenin  5 ± 0.50.04 ± 0.02—H═O—OH—OH—OHLuteolin1212 ± 45 3.24 ± 0.01—H═O—OH—OH—OH—OHGalangin18 ± 11.01 ± 0.03—OH═O—OH—OHFisetin1623 ± 1993.68 ± 0.03—OH═O—OH—OH—OHKaempferol1243 ± 99 1.84 ± 0.01—OH═O—OH—OH—OHQuercetin2383 ± 2583.27 ± 0.04—OH═O—OH—OH—OH—OHTamarixetin165 ± 201.14 ± 0.03—OH═O—OH—OH—OH—OMeRutin670 ± 413.18 ± 0.01—ORut*═O—OH—OH—OH—OHMyricetin14463 ± 17674.08 ± 0.01—OH═O—OH—OH—OH—OH—OHTri-Ome- 74 ± 141.06 ± 0.02—OH═O—OH—OH—OMe—OMe—OMeMyricetinDatiscetin22 ± 21.74 ± 0.02—OH═OMorin10134 ± 459 1.83 ± 0.01—OH═O—OH—OH—OH—OHVitamin E524 ± 482.14 ± 0.12—OH—OH—OHSecond order rate constants (k2)and reaction stoichiometries for the reduciton of galvinoxyl radical by flavonoids and vitamin E.*Rutin is quercetin-3-rutinoside. The compounds above the dotted line are based on the 2-H flavan system, while those below are Δ-2-flavan-4-ones.
TABLE 2−Eda-tocmyricetinControl AControl B9c9d9eMean182.78319.9996147.63062158.348236.525117.461121.74365.5291SEM8.602670.863786.60996353.912529.513979.0177510.0664ClogP12.0480.6370.3780.9561.9843.0424.19e*9f9g9g*9h9i*9jMean112.54646.187921.688919.11362.102132.9769107.849SEM14.23289.976870.510331.7618512.63679.4896711.2272ClogP3.975.1586.2165.9567.2748.47110.448
TABLE 3−Eda-tocmyricetinControl BControl CControl D9gMean182.782519.99965147.6306236.5249186.6221172.089921.68894SEM8.6026730.8637836.6099649.0765492.3936820.510328ClogP12.0480.6370.9560.4560.4566.216Control E11g1213g1415gMean206.032881.8186653.9825720.1401104.4307114.1373SEM10.9068810.011793.7222998.68617113.81451ClogP6.7676.2166.1365.7165.1875.716